Dopaminergic neurons control aspects of mood, cognition and movement, and are involved in the pathogenesis of a variety of brain disorders including drug addiction. A central regulator of dopaminergic neurotransmission is the dopamine (DA) transporter (DAT) that is expressed exclusively in dopaminergic neurons and is responsible for clearance of extracellular DA. Psychostimulants such as amphetamines and cocaine elicit dramatic behavioral phenotypes primarily by targeting DAT directly and increasing extracellular DA concentration. DA neurons are functionally heterogeneous and morphologically complex, with spatially separated somatodendritic and axonal domains, and dense axonal arborizations. Proper DAT distribution in the DA neuron is critical for the function of the dopaminergic system in its normal and diseased states. The principal goal of this proposal is to elucidate mechanisms by which the DAT-mediated substrate re- uptake is regulated in DA neurons by endocytosis, long-distance intraneuronal trafficking and membrane diffusion. We have developed and characterized a knock-in mouse that expresses an extracellular epitope (HA)-tagged DAT (HA-DAT). In this mice we used a combination of quantitative electron microscopy of intact brain and acute sagittal brain slices, a novel physiological ex vivo model of the intact DA neuron, to demonstrate distinct patterns of DAT trafficking in different regions of DA neurons. Using mechanistic analysis in cultured cells, we have identified molecular determinants in the DAT that are involved in its targeting to filopodia and its mobility in the plasma membrane, and novel sequence motifs that are important for constitutive and signal-induced DAT endocytosis. We propose that since intraneuronal distribution of DAT is regulated by the balance of vesicular traffic, cell surface retention and membrane mobility of DAT, these processes must be controlled differently by intracellular signaling in somatodendritic and axonal domains of neurons and in different areas of the striatum and midbrain. To test these hypotheses, we will: 1) Define kinetics and mechanisms of long-distance DAT trafficking between spatially separated axonal and somatodendritic domains of DA neurons; 2) Define routes, kinetics, and mechanisms of constitutive and signal- induced endocytosis of DAT in DA neurons; 3) Test whether brain region- and signal-specific DAT trafficking affects DAT substrate transport kinetics. Defining mechanisms of DAT regulation by trafficking in intact DA neurons will significantly advance our understanding of the cell biology and physiology of the DA system, and may suggest potential therapeutic options for treatment of DA pathologies such as drug addiction.